Alpha-lactalbumin, is a modifier protein of galactosyltransferase thereby promoting the transfer of galactose to glucose, resulting in a -1--4 glycosidic linkage and synthesis of lactose. It bears a structural homology with lysozyme, an enzyme catalyzing the hydrolysis of a -1--4 glycosidic linkage in polysaccharides. We have isolated clones carrying rat Alpha-LA genomic sequences from a bacteriophage Charon 4A/rat partial EcoRI genomic library. We have established the complete nucleotide sequence of the rat Alpha-lactalbumin gene carrying the coding and intervening sequences including its 5 feet-flanking region. Comparison of this gene structure with chicken egg white lysozyme gene shows a) both genes contain 3 introns at similar positions; b) the first three exons of the two genes show high nucleotide homologies and are of comparable lengths and c) the fourth exon of Alpha-lactalbumin, which codes for the amino acid residues essential for its interaction with galactosyltransferase, is markedly different from the corresponding exon of lysozyme. It is suggested that the 4th exon of Alpha-LA, coding for a new functional unit, might have replaced the DNA region of a primordial lysozyme gene and led to a protein with a new function.